Coenzyme Dependency

All type I BVMOs are strictly dependent on NADPH which delivers the electrons needed for catalysis. While the affinity for NADPH is high (KM 0.4-24 |imol L-1), type I BVMOs show no significant activity with NADH. The elucidation of the crystal structure of PAMO has revealed the molecular basis for this specificity. K336 appears to play a major role in binding of the 2'-phosphate of NADPH and is conserved in most type I BVMO sequences. Replacing this residue in HAPMO resulted in a drastically reduced affinity of NADPH while recognition of NADH improved [36]. The identification of residues recognizing the 2'-phosphate group enables structure-inspired enzyme engineering to create a BVMO accepting NADH. Such an enzyme mutagenesis approach will involve replacement of a number of residues, including K336. A BVMO which would be active with NADH is clearly advantageous for biocatalytic applications involving isolated enzyme as the non-phosphorylated cofactor is less expensive when compared with NADPH [47]. Engineering a BVMO which is active with NADH (and NADPH) might also prove to be valuable for conversions using whole cells as E. coli typically maintains high levels of NADH [48].

An alternative way to circumvent NADPH-dependent biocatalysis is to use artificial reducing agents. While for PAMO it has been shown that an artificial electron donor can be used [49], this approach has several significant disadvantages prohibiting applications. In general, artificial electron-donating catalysts/ agents are not as efficient as the natural coenzyme and often are costly. For PAMO it was also observed that binding of NADP+ is needed for efficient and enantiose-lective oxidations [49]. Electrochemical reduction has also been successfully applied to flavin-dependent monooxygenases [50]. Such an approach appears well suited for developing biosensors but is not (yet) applicable for synthetic purposes.

At the moment, the most effective method for in vitro regeneration of NAD(P) H is to couple the monooxygenase with an ancillary recycling enzyme. While for a long time glucose-6-phosphate dehydrogenase and formate dehydrogenase have been most popular as nicotinamide coenzyme recycling enzymes, several interesting alternative enzymes have been reported in the last years. Alcohol dehydro-genases from hyperthermophilic Archaea clearly are advantageous as recycling biocatalysts as they are (thermo)stable and tolerate organic solvents [47]. The use of an alcohol dehydrogenase in combination with a Baeyer-Villiger monooxygenase also makes it possible to perform a cascade reaction in which an alcohol is converted into the corresponding ester/lactone via formation of the ketone [51, 52].

Another attractive dehydrogenase for NAD(P)H recycling is phosphite dehydrogenase [53]. This enzyme is easily expressed in E. coli and is able to convert the inorganic substrate phosphite into phosphate while generating NADPH from NADP+. As the reaction is practically irreversible and phosphite can be bought as relatively inexpensive salt, this enzyme facilitates (cost-) efficient coenzyme regeneration.

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